7. Project summary. Protein?protein interactions (PPIs) regulate a plethora of fundamental biological processes and their misregulation or aberrance have been associated with a variety of diseases. Given that the chemical space covered by our current arsenal of small-molecule drugs only targets about 30% of known disease-relevant proteins, an incredible repertoire of compelling protein targets remains untapped. Therefore, the modulation of intra- and extracellular PPIs has recently emerged as one of the most exciting strategies towards next-generation therapeutics. The current state-of-the-art in drug discovery for inhibiting PPIs is monoclonal antibodies (mAbs) and a few promising large macrocyclic peptides. In contrast to typical binding pockets in proteins which are easily targeted by small-molecule drugs, the interfaces between proteins exhibit rather flat and large surface areas (800-2000 2) which are challenging to bind by small molecules. Obviously, peptides and peptidomimetics are well suited to mimic interfacial residues and protein epitopes, but their innate linearity and flexibility in solution result in low affinity to the protein target and renders these molecules vulnerable to proteolysis. To solve this issue, peptide stapling of helices, ?-sheets and ?-hairpins and peptide macrocyclization have been the most common and successful strategies for protein epitope mimicking (PEM). Following the 2014 report from Kritzer, suggesting that ~50% of PPIs and ?hot spot? contacts are embedded in non-regular secondary structures (e.g. loops) which cannot be mimicked by the current strategies, we have been inspired to develop new tools to synthesize small-molecule antibody mimics comprised of semi-flexible loops. This research proposes to develop a novel technology towards unnatural ?-hairpin designs (conformational rigidity, cell permeability and proteolytic stability) folded around a ?-sheet architecture and utilizing a fragment-based approach for crafting the loop pharmacophores and ?hot? contact residues (aim 1). As proof of principle, our rational drug-design approach will then be tested using synthetic bioactive ?-hairpins as inhibitors of PPIs (aim 2). The innovation of this technology is intended to complement the existing PEM strategies by synthesizing and crafting ?hot loops? that mimic the native hypervariable loops of mAbs onto some rigid ?-sheets composed of unique peptoid building blocks. These studies will allow us to delineate a protocol to discover protein epitopes embedded in loops and ultimately create a broadly applicable technology in drug discovery for PPI inhibitors that will enable Medicinal Chemists to tackle targets that have been previously deemed undruggable (aim 2). Such transformative technology could potentially be a game-changer in the study of novel medium-sized peptide drugs able not only to bind to extracellular receptors, but also to target intracellular proteins which cannot be engaged with mAbs. As a result, a large number of biologically-relevant targets from various diseases could become more druggable and offer new possibilities for the development of personalized medicines.